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Coamplification of multiple segments of chromosome 2, including an MYCN-bearing segment, was examined in 2 cancer cell lines, NCI-H69 (lung cancer) and IMR-32 (neuroblastoma). High-resolution array-CGH analysis revealed 13 and 6 highly amplified segments located at different sites in chromosome 2 in NCI-H69 and IMR-32, respectively. FISH analysis demonstrated that these segments were co-localized in double minutes in NCI-H69 and in homogeneously staining regions in IMR-32. Connectivity of the segments was determined by a PCR assay using designed primer sets. It was found that all the segments were connected to each other irrespective of their order and orientation against the genome sequence, and a single chain-like cluster was configured in both cell lines. Such patchwork structures of the amplicons suggest the possibility that massive genomic rearrangements, explained by the single catastrophic event model, are involved in the formation of the amplicons, enabling the coamplification of different chromosomal regions including the MYCN locus. The model comprises massive fragmentation of chromosomes and random rejoining of the fragments., (Copyright © 2011 S. Karger AG, Basel.)

Lung cancer sublines No15-80-1 and No15-80-6 were selected by treatment of cell line NCI-H460 with paclitaxel at stepwise increasing concentrations from 50 nmol/L to 800 nmol/L. The two sublines exhibited amplifications of the ABCB1 region (previously MDR1) with different copy number profiles, but shared a common amplification pattern, which has been observed in amplification mediated by the breakage-fusion-bridge (BFB) cycle. Sequence analysis of the distal ends of the amplified regions, which were probably generated in a break-and-fusion of the initial round of the BFB cycle, revealed a head-to-head fused sequence of chromosome 7. The sequence was identical in the two sublines. A short sequence of 200bp derived from chromosome 2 was incorporated, suggesting translocation between chromosomes 2 and 7. The copy number of the short sequence was comparable to that of the neighboring sequence, suggesting coamplification. The timing of the occurrence of the putative translocation and the initiation of BFB-cycle-driven amplification during the stepwise selection were determined by using the unique junction sequences specific to these events as indicators. The results demonstrated that the translocation occurred at the step of 100 nmol/L treatment and the BFB cycle initiated in the step of 400 nmol/L-treatment. It is likely that the translocation, preceding amplification by several selection steps, activated ABCB1 gene expression. The diversity in amplification profiles between the two sublines was generated by the separately operating BFB cycles, after an initial break-and-fusion that probably occurred in a single cell.

A multidrug-resistant lung cancer cell line PTX250, established by treatment with the anti-cancer drug paclitaxel, has been demonstrated to have an increased copy number in the 7q21.12 region including the MDR1/ABCB1 gene. The amplicon is 2.7 megabases in size, and the copy number increase is 11-fold compared with the parental cell line. Here, we examined the amplicon structure and determined nucleotide sequences at both junctions of the amplicon. Fluorescence in situ hybridization analysis using an MDR1 probe demonstrated a cluster of fluorescent signals at the chromosomal end, suggesting an intra-chromosomal amplification. DNA fragments of both junctions were cloned and sequenced. The distal junction was a head-to-head fusion with a 4-base pair (bp) overlap separated by an asymmetric sequence of 1,265 bp, and the proximal junction was a tail-to-tail fusion with a 2-bp overlap intervened by an asymmetric sequence of 2,203 bp. These results suggest that the amplicon has a large palindromic structure with an asymmetric sequence and has been amplified through the breakage-fusion-bridge cycle. Specific sequences, which might be related to the occurrence of double-strand-breakages, were found at or near the junctions of the amplicon -- an inverted repeat in the distal junction and a highly AT-rich region near the proximal junction.

A paclitaxel-resistant subline was generated from the non-small lung cancer cell line NCI-H460 by stepwise selection in paclitaxel from 0.032 to 250 nmol/L. The resulting subline, designated NCI-H460/PTX250, showed 792-fold resistance against paclitaxel compared with the parental cell line NCI-H460. The chemosensitivity analysis revealed the cross-resistance phenotype against various anticancer drugs including docetaxel, vinblastine, and doxorubicin, but not against camptotecin, cisplatin, and 5-fluorouracil. The addition of 5 mumol/L verapamil or reversin 121 reversed the resistance against paclitaxel, vinblastine, and doxorubicin. The gene expression profile, examined using oligonucleotide microarrays, demonstrated that the expression of 332 and 342 genes was significantly increased and decreased, respectively, in NCI-H460/PTX250 compared with NCI-H460. The most highly upregulated gene was MDR1/ABCB1 with a 1,092-fold increase. The overexpression was confirmed at the protein level by Western blot and flow cytometry analyses. The copy number profile, examined using microarray-based comparative genomic hybridization, revealed amplification of the q11.21 approximately q21.12 region on chromosome 7. In particular, the entire q21.12 region displayed 11- to 13-fold higher copy number in NCI-H460/PTX250 than in NCI-H460. Most of the genes within the region were highly expressed, and the increased expression of these genes could be explained by the amplification in the gene copy number. However, the increase in MDR1/ABCB1 expression greatly exceeded the genomic copy number increase of the gene, suggesting the existence of one or more additional factors, such as transcriptional enhancement or mRNA stabilization, associated with the elevated MDR1/ABCB1 expression. In conclusion, both chromosomal region-specific copy number amplification and gene-specific activation are probably involved in the overexpression of MDR1/ABCB1, resulting in acquisition of the drug resistance phenotype in NCI-H460/PTX250.

Alu elements are short, ∼300-bp stretches of DNA and are the most abundant repetitive elements in the human genome. A large number of chromosomal rearrangements mediated by Alu-Alu recombination have been reported in germline cells, but only a few in somatic cells. Cancer development is frequently accompanied by various chromosomal rearrangements including gene amplification. To explore an involvement of Alu-Alu fusion in gene amplification events, we determined 20 junction site sequences of 5 highly amplified regions in 4 cancer cell lines. The amplified regions exhibited a common copy number profile: a stair-like increase with multiple segments, which is implicated in the breakage-fusion-bridge (BFB) cycle-mediated amplification. All of the sequences determined were characterized as head-to-head or tail-to-tail fusion of sequences separated by 1-5 kb in the genome sequence. Of these, 4 junction site sequences were identified as Alu-Alu fusions between inverted, paired Alu elements with relatively long overlapping sequences of 17, 21, 22, and 24 bp. Together with genome mapping data of Alu elements, these findings suggest that when breakages occur at or near inverted, paired Alu elements in the process of BFB cycle-mediated amplification, sequence homology of Alu elements is frequently used to repair the broken ends. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]